DNA damage can result in mutation, and this is a primary mechanism by which cancers arise. These events have also been implicated in diseases such as atherosclerosis, and processes such as aging. Therefore, there is an important need for sensitive methods which are capable of identifying chemical or physical agents that can mutate DNA. Given the tremendous cost of long-term chronic studies such as 2-year carcinogenicity tests, short- and medium-term systems for predicting DNA reactivity play a vital role in tumorigenic agent identification.
Although sensitive assays for measuring in vitro mutation exist (e.g., Salmonella reverse mutation test, mouse lymphoma forward mutation assay), in vivo methods are needed to form a more complete understanding of risk. Thus, while there are National Institutes of Health (NIH) directives to reduce animal usage for toxicity testing purposes, it is well appreciated that whole animal systems are necessary for faithful incorporation of variables such as toxicant deposition, metabolism, and elimination. Because the in vivo system proposed herein is based on blood sampling and therefore does not require sacrifice, it can easily be integrated into on-going acute, subacute, or subchronic toxicology studies. This approach would help minimize animal usage, as the number of dedicated experiments conducted to assess genotoxicity is reduced. Furthermore, since it should be possible to extend the method beyond laboratory rodents to man, it could play an important role in studying new drugs during clinical trials, or for myriad other human biomonitoring applications.
Some of the more widely utilized assays for studying in vivo mutation are listed in Table I below along with the assay of the present invention. While some are based on colony formation and therefore require time-consuming tissue culture work after target cells have been harvested, others require expensive breeding programs to supply rodents with a specific genotype.
TABLE IOverview of in vivo Mutation AssaysSpecialCultureTarget GeneRequirementsTarget CellsWorkEnumerationhprtCompatible with allBlood (or spleen)YesColonymammalslymphocytesformationthymidineRequires tk+/− miceBlood (or spleen)YesColonykinaselymphocytesformationGlycophorin AHeterozygous humansBlood RBCsNoFlow(M-N blood antigens)cytometryTransgenic lacZProprietary rodentsAnyYes (bacteriaPlaqueor lacI(e.g., Muta ™ Mouse,and phage)formationBigBlue ® Mouse orRat)pig-a*Compatible with allBlood RBCs (andNoFlowmammalsother blood cells)cytometryAbbreviations:hprt = hypoxanthine phosphorylribosyl transferase;tk+/− = thymidine kinase heterozygote*pig-a is the target of the present invention; it is compared here based on the manner of detection described in the present application.
As Table I suggests, the invention described herein is not the first to quantitatively measure in vivo mutation. The novelty and advantages of the present invention stem from careful attention to the choice of gene locus and the target cells. Importantly, these mutation data will be available without the need for time and resource-intensive tissue culture work, or the use of costly transgenic animals. Furthermore, given the compatibility of enumerating the mutant phenotype via flow cytometric analysis, the assay of the present invention is endowed with a high throughput capacity. The methodology of the present invention has other advantages relative to the current state-of-the-art. These advantages include: compatibility with any mammalian species, easy integration into other studies, and conceivably higher relevance for the reporter gene, which is endogenous and transcribed (as compared to transgenes which are non-transcribed, have high G-C content, and are extensively methylated).
The pig-a gene is located on the X-chromosome. This is a highly desirable feature, since one functional copy means that only a single mutational event is necessary to produce a phenotype which can be readily detected. More specifically, the pig-a gene product is essential for the biosynthesis of glycosyl-phosphatidylinositol (GPI) anchors. Mutations giving rise to non-functional pig-a product result in the absence or the reduced membrane expression of GPI-linked proteins in peripheral blood cells. See FIG. 1.
Paroxysmal Nocturnal Hemoglobinuria (PNH) is a genetic disorder that affects 1 to 10 per million individuals. The molecular basis of PNH is a somatic pig-a gene mutation within a bone marrow stem cell. PNH usually affects erythrocytes, granulocytes and monocytes. Occasionally, only the erythrocytes, or the granulocytes and monocytes, are affected. In a minority of cases the lymphocyte lineage is also affected, and only a few rare case reports have documented the lymphocytes to be the only cell lineage affected. Several GPI-linked proteins, especially CD59 and CD55, have been studied intensely. In fact, flow cytometry-based techniques which measure the frequency of CD59 and/or CD55 deficient red blood cells are replacing the traditional HAM test for PNH diagnosis. Even so, it is important to recognize that since clinically significant disease requires a relatively large fraction of cells to exhibit GPI-anchor deficiency, these PNH diagnostic assays have not required the degree of accuracy that a rare-event/mutation scoring system will require. Thus, the repurposing of pig-a gene mutation measurements for evaluating genotoxicity, as opposed to diagnosing PNH disease, requires the high throughput and reliability characteristics of the present invention.
The present invention is directed to overcoming these and other deficiencies in the art.